Nauseef W M, McCormick S J, Goedken M
Inflammation Program and Departments of Medicine, University of Iowa and Veterans Affairs Medical Center, Iowa City, Iowa 52242, USA.
J Biol Chem. 1998 Mar 20;273(12):7107-11. doi: 10.1074/jbc.273.12.7107.
Myeloperoxidase (MPO) is a neutrophil lysosomal hemeprotein essential for optimal oxygen-dependent microbicidal activity. We have demonstrated previously that calreticulin, a luminal endoplasmic reticulum protein, functions as a molecular chaperone during myeloperoxidase biosynthesis, associating reversibly with the heme-free precursor apopro-MPO. Because the membrane-bound endoplasmic reticulum protein calnexin is structurally and functionally related to calreticulin, we assessed the role of calnexin in myeloperoxidase biosynthesis. Like calreticulin, calnexin coprecipitated exclusively with glycosylated MPO precursors and with apopro-MPO but, in contrast to calreticulin, also with the enzymatically active, heme-containing precursor pro-MPO. To determine if calnexin participated in heme insertion into MPO, we compared the kinetics of chaperone association with MPO precursors using stable transfectants expressing cDNA encoding wild type MPO or mutated forms that do not acquire heme. Transfectants expressing mutant cDNA had prolonged association of MPO-related precursors with calreticulin and especially with calnexin. These studies demonstrate that 1) both calreticulin and calnexin associated with glycosylated apopro-MPO; 2) only calnexin associated selectively with the enzymatically active, heme-containing precursor pro-MPO; and 3) mutants unable to incorporate heme had prolonged association with calnexin. These findings represent the first evidence of a specialized role for calnexin in facilitating protein maturation in the endoplasmic reticulum of myeloid cells.
髓过氧化物酶（MPO）是一种中性粒细胞溶酶体血红素蛋白，对最佳氧依赖性杀菌活性至关重要。我们之前已经证明，内质网腔蛋白钙网蛋白在髓过氧化物酶生物合成过程中作为分子伴侣发挥作用，与无血红素的前体脱辅基MPO可逆性结合。由于膜结合内质网蛋白钙连蛋白在结构和功能上与钙网蛋白相关，我们评估了钙连蛋白在髓过氧化物酶生物合成中的作用。与钙网蛋白一样，钙连蛋白仅与糖基化的MPO前体和脱辅基MPO共沉淀，但与钙网蛋白不同的是，它也与具有酶活性的含血红素前体前MPO共沉淀。为了确定钙连蛋白是否参与血红素插入MPO，我们使用表达编码野生型MPO或不获取血红素的突变形式的cDNA的稳定转染细胞，比较了伴侣蛋白与MPO前体结合的动力学。表达突变cDNA的转染细胞中，MPO相关前体与钙网蛋白尤其是与钙连蛋白的结合时间延长。这些研究表明：1）钙网蛋白和钙连蛋白都与糖基化的脱辅基MPO结合；2）只有钙连蛋白选择性地与具有酶活性的含血红素前体前MPO结合；3）无法掺入血红素的突变体与钙连蛋白的结合时间延长。这些发现首次证明了钙连蛋白在促进髓样细胞内质网中蛋白质成熟方面具有特殊作用。
